Method for analyzing t lymphocytes with the aid of t lymphocyte receptors of an organism

ABSTRACT

The method for analyzing the activation and motivation of T cells by analysis of T lymphocyte receptors of an organism consists in the following: determination of the number of transcripts for the gene Vβ in order to obtain first data; amplification of the region CDR3 of the T lymphocyte receptors for each of the Vβ genes associated with the organism; separation of the different lengths of the CDR3 of the T lymphocyte receptors for each of the Vβ genes associated with the organism and estimation of the proportion of transcripts of each length of the CDR3 in each Vβ family in order to obtain second data; representation of the first and second data according to the Vβ genes and the length of the regions CDR3 in a diagram with 4 variables.

The invention relates to the study of the immune system of organismssuch as humans or animals, in particular to the analysis of activated ornonactivated T lymphocytes of an organism though studying its receptor.

Immune responses can be classified in two major categories: naturalresponses and adaptive (or specific) responses. The cells involved inthe natural response use primitive and non-specific recognition systems.This type of response therefore represents the first line of defenseagainst infections and predominates at the initial stages of infection.When the natural immunity is ineffective, adaptive immunity, whichinvolves specific responses adapted to each microorganism, is set up.Lymphocytes form the basis of this adaptive recognition.

Two types of lymphocyte exist: B lymphocytes, responsible for humoralimmunity (antibody production) and T lymphocytes, responsible forcellular immunity. The latter play a central role in biology. Theyrecognize peptides derived from the intracellular degradation ofantigens, but only when these peptides are associated with moleculescarried by antigen-presenting cells, which molecules are called MajorHistocompatibility Complex or MHC (also called Human Leukocyte Antigenor HLA) molecules, carried by antigen-presenting cells. For thisfunction, T lymphocytes use a specific receptor called T lymphocyteantigen receptor or TCR.

The T cell receptor (TCR) is a heterodimer consisting of two chains: αand β. We are interested here only in the β-chain which comprises the Vβgenes (24 in humans), Dβ genes (2 in humans), Jβ genes (12 in humans)and Cβ genes (2 in humans), which are initially separated on the DNA.One of the Vβ genes becomes linked randomly to one of the Dβ, Jβ and Cβgenes so as to form a functional receptor. Thus, a large number ofdifferent TCRs can theoretically be generated (approximately 25×10⁶different TCRs). An encounter with an antigen leads to the selection ofT lymphocytes bearing a specific TCR consisting of a particular Vβ-Dβ-Jβassembly. Recognition of the antigen occurs mainly at the level of theCDR3 (Complementary Determining Region 3) hypervariable region which isformed by the rearrangement of the Vβ, Dβ and Jβ genes and which variesin length. In a healthy individual, the transcripts encoding any Vβfamily normally use all the possible lengths of the CDR3.

Up until now, analysis of the T cell receptor has mainly been carriedout “qualitatively” (i.e. the search for a possible selection) by virtueof methods such as that used by the software known under the trademarkImmunoscope® and which studies this receptor in terms of the lengthdistribution for the CDR3 region, which region is thought to be the mosthighly involved in peptide recognition.

After amplification of this CDR3 region, of varying length, by virtue ofa Cβ primer and a Vβ primer, the Immunoscope software provides theresults in the form of peaks, each profile consisting of 7 to 11 peaks,i.e. of 7 to 11 different lengths for the CDR3, each separated by 3nucleotides. Thus, when all the CDR3 lengths are represented in a givenVβ family, a Gaussian profile, characteristic of a polyclonal response,is observed. This is, for example, what is generally observed in ahealthy individual. On the other hand, a CDR3 length can be representedin the majority subsequent to a particular activation which has selectedparticular T lymphocyte clones, and an oligo- or monoclonal “alteration”of the CDR3 region length distribution is then observed, the profilesthen losing their Gaussian nature

One means for comparing the profile of a sample relative to the controlprofile is to use the calculation described in the article by G.Gorochov (Nat. Med., 1998, 4(2):215) which calculates the differencebetween the area under each peak of the control profile and the areaunder each peak of the profile of the sample. Percentages of alterationrelative to the Gaussian profile are thus obtained, and the results arerepresented in the form of three-dimensional structures which make itpossible to visualize the alterations associated with all the Vβfamilies of a given sample on the same graph. These graphs, commonlycalled Reperturb, appear to be flat when the repertoire is not alteredand perturbed when the repertoire is altered.

Despite their advantage, these methods for qualitatively analyzing the Tcell receptor do not make it possible to precisely determine the amountof Vβ mRNA affected by a selection process (and therefore indirectly thesize of the pool of T cells using a given Vβ rearrangement) andtherefore have a considerable limitation in that they are incapable ofplacing the abnormalities observed in order of size of representation.

By way of example, FIG. 2 represents a Gaussian profile and FIG. 1 amonoclonal expansion obtained by Immunoscope®. Using the Reperturbmethod, four peaks, each corresponding to a Vβ family exhibiting aclonal expansion, appear in FIG. 3. Despite its value, this qualitativeanalysis alone does not make it possible to determine whether the TCRsexhibiting these expansions involve a large fraction of the Vβtranscriptome or whether they represent only a minority among the T cellpool. For example, in FIG. 3, it is not known whether the T cellsbearing one of the 4 clonal expansions is represented to a greaterextent than another among the total T cell population, and is thereforemore particularly involved in the immune reaction studied. On the otherhand, the graph remains flat when all the Vβ families exhibit a Gaussiandistribution for CDR3 lengths, as shown in FIG. 4. This flat profile maybe due:

-   -   to a lack of response (no T lymphocyte is involved),    -   to a polyclonal response due to the activation of a large number        of T clones, this activation masking the presence of potential        clonal expansions,    -   or to the activation of T cells without modification of the CDR3        length distribution. This is, for example, the case of a profile        observed after stimulation with polyclonal ligands (anti-CD3        antibody, Concanavalin A, PHA, etc.) or with superantigens.

Qualitative analysis alone does not make it possible to differentiatebetween these three hypotheses.

The applicant was the first to use the Immunoscope® method to studyallograft rejection and tolerance or, more recently, xenograft rejectionand tolerance. However, although these studies were carried out oncongenic rat lines (a combination which is much more “simplified” thanthe allogenic combination is), they revealed alterations of the“private” type (specific for an individual) in most cases, while“public” alterations (same length and same sequence for the CDR3 foundin several animals) are extremely rare events. Due to this dominant“private” (and therefore apparently chaotic) profile and to the highcross-reactivity of the TCR, no reproducible information on thephysiology of allorecognition can be obtained. Based on theseobservations and this finding, it appears that a combined analysis ofthe qualitative (“private”/“public”) alterations and of the quantitativeestimation of the pool of T cells involved in these alterations (numberof VP transcripts for each profile altered) is the only means ofapproaching T lymphocyte response kinetics.

Thus, our ability to evaluate and to study the dynamics and the size ofthe T lymphocyte response in vivo is very limited. Analysis of the TCRrepertoire is an indirect reflection of the state of mobilization and ofactivation of T cells in a given biological situation. However, theconsiderable cross-reactivity of the TCR and the great diversity of thepeptides processed make it difficult to use the qualitative alterationsof the Vβ repertoire taken alone (for example according to theImmunoscope method) as a marker for precisely identifying and placing inorder of representation the population of T cells involved in a compleximmune reaction, even if the study of the repertoire is in terms of thesequences and the variations in length of the CDR3 region. Despite theirvalue, the methods previously known for analyzing the TCR did nottherefore make it possible to obtain a precise determination of the mRNApool of T lymphocytes (relative to the overall Cβ transcriptome) using arearrangement of given Vβ chains. In addition, accumulation of Vβtranscripts is a different parameter from the expression of thecorresponding proteins at the surface of T cells since these proteinsmay be down-regulated after interaction with the antigen.

One of the aims of the invention is to provide a novel method ofanalysis which makes it possible to add a quantitative dimension, aparameter which is essential to analyzing repertoire alterations.

For the purpose of achieving this aim, we propose, according to theinvention, a method for analyzing T lymphocyte receptors of an organism,which comprises the steps consisting in:

-   -   determining the number of transcripts for each Vβ gene, so as to        obtain first data. This number of transcripts may then be        represented, for example, in the form of crude values (number of        transcripts for each Vβ family), in the form of a ratio between        this number of transcripts and the number of transcripts of a        reference gene, for example the HPRT (hypoxanthine        phosphoribosyl transferase) gene, the latter being a gene which        is present in all cells and which makes it possible to        standardize the data, in the form of a percentage of each Vβ        transcript among the sum of Vβ transcripts, or in the form of a        measurement of the variation between the number of transcripts        in a given Vβ family of a given sample and the number of        transcripts of the same Vβ family of a reference sample;    -   amplifying the CDR3 region of the T lymphocyte receptors for        each of the Vβ genes associated with the organism;    -   separating the various lengths of the CDR3 of the T lymphocyte        receptors for each of the Vβ genes associated with the organism        and estimating the proportion of transcripts for each length of        the CDR3 in each Vβ family, so as to obtain second data;    -   representing, in a four-variable diagram, the first and second        data as a function of the Vβ genes and of the length of the CDR3        regions.

Thus, the invention makes it possible to follow with precision theexistence and the quantitative hierarchy of abnormalities in the lengthdistribution for the CDR3 region during the initiation, kinetics,expansion, memory and degree of epitope spreading in a complex immuneresponse situation (for example a pathological context), due to the factthat the two parameters (profile of alteration of the lengthdistribution for the CDR3 region of the TCR compared to the restingsituation, and quantitative evaluation of the Vβ transcripts in the poolof T cells involved) can be visualized overall, simultaneously, on thediagram.

The invention therefore provides an integrated view of the alterationsin the TCR, instead of only an individual description of the alterationsof each Vβ family. It is more suitable for understanding the fundamentaland applied immunological events involving a T lymphocyte response(infectious diseases, auto-immune diseases, cancers, transplants, etc.).The presentation of the results is also at the same time complete,didactic and easy to understand.

The invention proposes a novel strategy for evaluating the immuneresponse in vivo, preferably based on a computer-assisted integration ofthe alterations of the Vβ transcriptome of activated Tαβ0 lymphocytes.

The method of the invention combines analysis, obtained for example withthe Reperturb and Immunoscope® programs, of the alteration in CDR3length (in a given Vβ family), compared with the resting Gaussianprofile, with a quantitative measurement of each Vβ transcript byquantitative PCR (for example according to the method known as TaqMan)for each Vβ family and therefore of the transcripts for all the alteredTCR signals. A factorial analysis is used to define the coherence of Vβuse and of alteration thereof. The invention includes a graphicexpression program which allows a general and, for example,three-dimensional view of the hierarchy of the alterations and thereforeof the state of activation of the reactive T cells.

The method according to the invention may also have at least any one ofthe following characteristics:

-   -   at least one of the variables is represented without being        associated with a dimension in space;    -   at least one of the variables is represented by differences in        local appearance of the diagram;    -   the differences in local appearance are color differences;    -   the variable which is not associated with a dimension in space        corresponds to the second data;    -   the first data are represented in the vertical direction;    -   the predetermined values correspond, for each Vβ gene, to a        Gaussian distribution of the proportions of transcripts        corresponding to the CDR3 regions associated with this gene;    -   it comprises, after determination of the first and second data,        the steps consisting in choosing a Vβ gene as a function of the        results from the first two steps, and in extracting from the        total T cell population the T cells bearing a TCR consisting of        this preselected Vβ family; and    -   at least some of the steps are carried out in an automated        fashion.

A computer program for analyzing T lymphocyte receptors of an organism,able to control the implementation of at least the representation stepof the method according to the invention is also provided for accordingto the invention.

Also provided for according to the invention is an installation foranalyzing T lymphocyte receptors of an organism, comprising:

-   -   automated means for measuring the amounts of each Vβ gene and,        optionally, the ratio of these amounts to predetermined values,        so as to obtain first data;    -   automated means for amplifying the CDR3 region for each of the        Vβ genes for the T lymphocyte receptors associated with the        organism, and for measuring, for each Vβ gene, the relative        amount of each CDR3 region with respect to all the CDR3 regions        associated with a Vβ gene, so as to obtain second data; and    -   automated means for representing, in a four-variable diagram,        the first and second data as a function of the Vβ genes and of        the length of the CDR3 regions. This diagram allows a simplified        and overall vision of the complex process of T cell mobilization        during an immune reaction.

Finally, the invention provides for an information medium exhibiting adiagram analyzing the T lymphocyte receptors of an organism, comprisingthe following four variables:

-   -   the Vβ genes associated with the organism;    -   the length of the CDR3 regions of the T lymphocyte receptors;    -   first values representing the amount of transcripts of each Vβ        gene in the form of relative or absolute amount;    -   second data representing, for each of the lengths of the CDR3        region in each Vβ gene, the difference between the proportion of        transcripts corresponding to a CDR3 region of a Vβ gene in a        given sample compared to the transcripts corresponding to the        same CDR3 region of the same Vβ gene in a reference sample which        is a sample exhibiting a Gaussian distribution for CDR3 lengths.

The invention may be the subject of many applications, for example inthe following immunological fields:

-   -   monitoring the progression of various pathological conditions        over time, such as cancers, viral diseases (AIDS, EBV infection,        etc.), autoimmune diseases, etc.;    -   monitoring the effect of various treatments (immunotherapy,        etc.);    -   studying the mechanisms of the immune response in various        diseases and/or subsequent to various therapies;    -   studying the mechanisms of rejection and tolerance in        transplantation;    -   diagnosing a “memory state”.

Some examples of applications are proposed more precisely below:

-   -   monitoring the progression of the disease and searching for a        possible correlation between the regression or the worsening of        the disease and the size of the “spreading”, which corresponds        to the fact that the T clone selection will evolve over time due        to the recognition of novel antigenic structures by molecular        mimicking;    -   monitoring the evolution, over time, of the T cell repertoire in        patients who may or may not be exhibiting a continuous        regression of the disease after immunotherapy. The invention        will make it possible to monitor both the effect of the therapy        and the development of the immune response against the disease;    -   detecting specific markers associated with the progression of        the disease. For example, an increase in clonal alterations, an        increase in the number of Vβ families exhibiting particular        alterations and/or a significant increase in Vβ messengers in        the blood, taken over considerable periods of time, may make it        possible to study the “epitope spreading”. Such differences will        make it possible to distinguish the T responses before and after        a treatment from the responses more highly involved in the        regression of the disease due to the therapy;    -   distinguishing between early immune processes and those which        have been set up for a long time in the case of autoimmune        diseases for example, and between the immune processes involved        in various forms of the same disease;    -   comparing the graphic representations provided via the invention        with the results obtained with conventional techniques of        diagnosis and of immunoanalysis carried out by other methods        (for example: monitoring of T cells by tetramers, monitoring of        the production of cytokines by the method known as Elispot) and        possibility of correlating the evolution of the appearance and        disappearance of T clones over time with the clinical        progression of the patient, so as to obtain important        information on the symptomatology and the immune processes        involved in the disease.    -   Various modifications may be observed after treatment of a        disease (disappearance of clones, modification of quantitative        values, modification of overall profile topology, etc.). These        modifications may indicate a down-regulation, the deletion of        autoreactive clones or else an evolution of the T cells        involved. This symptomatology may vary according to the strategy        used. Specifically, some agents can induce apoptosis of        autoreactive cells, while cytotoxic drugs such as cyclosporin or        mitoxantrone can cause aspecific modifications. Theoretically,        the profiles for the patients obtained using the invention might        therefore influence the decision regarding treatment. The        invention may also be useful for establishing criteria for        tolerance in patients apparently lacking clinical symptoms;    -   possibility of identifying, peripherally, specific        representations of certain diseases. Similarly, the possibility        of superimposing the graphs obtained from the blood and those        observed in the transplant is envisioned (for example for        detecting signs of chronic rejection via a blood sample);    -   The analysis according to the invention, on samples from        patients, may make it possible to define a new parameter for        selecting epitopes which are candidates for a more effective        immunotherapy (antitumor immunotherapy);    -   isolation of T cells revealed by the combination of the first        and second data as being particularly involved in the immune        reaction studied. An analysis of the specificity and of the        functionality of these isolated T cells may then be carried out.        This identification then isolation of T cells carrying a        particular TCR makes it possible to carry out functional or        phenotypic studies for example, not on the total T cell        population but only on the T cells involved in the immune        reaction studied. This improved “gene searching” approach will        make it possible to identify novel strategies for immunotherapy.        By way of example, see FIG. 14.

The stakes involved for many immunizing procedures are to develop a Tresponse and therefore to measure it. In this respect, the inventionrepresents a novel, potentially very useful, addition. Furthermore,studying the topology of the repertoires represented may be importantfor detecting the specific “signatures” of a pathological process in anindividual. Specifically, the invention can be used, for example, toanalyze the progression of the pathological process in patients and/orto influence the decision regarding a treatment.

In the field of pharmaceutical and medical research, the invention willbe of use for understanding and monitoring pathological conditionsand/or treatments in various fields such as:

-   -   virology;    -   cancerology;    -   autoimmune diseases;    -   treatment of allergies (monitoring of desensitizations);    -   transplantation of organs, of tissues or of marrow, anticipation        of the occurrence of acute or chronic rejection, search for        regulating clones in transplant patients in order to determine        whether these clones are liable to be tolerant with respect to        their transplant in the absence of any immunosuppressor        treatment, etc.

In medical research, the invention will be of use, for example, as aclinical tool to aid with diagnosis (viral diseases, autoimmunediseases, cancer) and for monitoring therapies (vaccines, celltherapies, gene therapies).

The invention can be used on animals in the context of research projects(animal tests, preclinical analyses, etc.) and on humans in the contextof treatment assessment.

Other characteristics and advantages of the invention will emergefurther in the following description of several preferred embodimentsgiven by way of nonlimiting examples, with reference to the attacheddrawings:

FIGS. 1 to 8 are diagrams relating to the study of T lymphocytes, andobtained with techniques of the prior art;

FIGS. 9 and 11 to 13 are diagrams obtained with the method according tothe invention;

FIG. 10 is a diagrammatic representation of the means for implementingthe invention and of the medium obtained; and

FIG. 14 represents an example of sorting. In this example, a TcLandscapeprofile was obtained from a patient suffering from multiple sclerosis.On the graph, it is observed that the Vβ17 family is altered and thatthe number of Vβ17 transcripts is large. The T cells bearing a TCRcomposed of Vβ17 genes were isolated by flow cytometry and functionalstudies were carried out on these isolated cells. Particularly hightranscription of the cytokines of I1-6, I1-8 and TNFα is observed inthese cells. This accumulation is not observed in the T cells bearing aVβ17+ TCR from a healthy individual. Furthermore, these Vβ17+ cellsisolated from the patient exhibit an activated phenotype.

EXAMPLES

A first embodiment of the method according to the invention will bedescribed in the context of the cellular response inxenotransplantation.

In transplantation, T cells can recognize the antigenic peptidespresented by the presenting cells of the recipient (indirect pathway) orof the donor (direct pathway). Since the direct presentation pathway isparticularly involved during acute rejection, the inventors studied theimpact of this type of recognition on the TCRβ repertoire. By virtue ofthe invention, which makes it possible to add a quantitative dimensionto the qualitative alterations in the length distribution for the CDR3region, it appeared that this distribution was not altered in the directrecognition, although the T cells are selected as a function of their Vβsegment. In addition, by following the regulation of expression of theTCR as a marker of activation, it is shown that a large proportion ofnaïve T cells can recognize “foreign” presenting cells, a phenomenonwhich makes it possible to understand more clearly the magnitude of thedirect response and its involvement in acute rejection.

Analysis of the TCR repertoire makes it possible to evaluate thediversity of a T lymphocyte population and to monitor the evolutionthereof under various experimental conditions or during the progressionof a given pathological condition. It is thus possible to demonstraterestrictions (absence of certain Vβ families) or alterations in therepertoire (modification of the CDR3 length distribution in a Vβfamily).

Analysis of the TCR receptor makes it possible to evaluate the diversityof a T lymphocyte population and to monitor the evolution thereof undervarious experimental conditions or during the progression of a givenpathological condition. It is thus possible to demonstrate restrictions(absence of certain Vβ families) or alterations in the repertoire(modification of the CDR3 length distribution in a Vβ family).

Various techniques have been developed in order to analyze the CDR3region and/or to quantify the variations in the repertoire at the levelof the Vβ chain of the TCR. Currently, quantitative analysis of the TCRcan be carried out by various methods: the RNAse Protection Assay, whichhas the disadvantage of being technically difficult, quantification froma competitive PCR or CD3, which does not distinguish between the variousVβ families, and anti-Vβ monoclonal antibodies. For quantitativeanalysis, many monoclonal antibodies directed against the variousvariable regions of the TCRαβ are available in humans and in mice andallow analysis of expression at the protein level. However, in rats,only three antibodies are available and effective, which limits thevalue of this method. In addition, analysis only at the protein leveldoes not take into account the selection events occurring in the CDR3region, which is the basis of the specificity of the antigenrecognition. In addition, the percentage expression of a Vβ protein isnot representative of the corresponding Vβ transcript percentage. Infact, such a quantification with antibodies runs the risk ofunderestimating the number of TCRs since they are internalized afteractivation. Other techniques, based on PCR amplification of the variableregions of the TCR using specific primers, make it possible to analyzethe repertoire from genomic DNA. The amplification products thusobtained can be analyzed by autoradiography, cloned and sequenced.

The qualitative analysis of the TCR β-chain (second data) is based onstudying the length distribution for the CDR3 hypervariable region ofthe TCR in each Vβ family of a given T lymphocyte population. A pool ofresting T cells is characterized by a Gaussian distribution of thevarious lengths of the CDR3 in each Vβ family. The mobilization ofspecific T clones subsequent to recognition of an antigen is accompaniedby the preferential use of certain TCRs composed of a Vβ family andhaving a particular CDR3 length.

The principle of the Immunoscope® technique consists of anamplification, by PCR, of the various CDR3 regions using a common 3′primer placed in the Cβ segment and a 5′ primer specific for each Vβfamily (see sequence listing). The products amplified are then subjectedto an elongation step carried out with a second Cβ primer, labeled witha fluorophore. The fluorescent amplification products are separated as afunction of their length by migration on a polyacrylamide gel. Themigration profile is analyzed with a DNA sequencer coupled to theImmunoscope® software. An image of the CDR3 length distribution is thusobtained, with reference to FIG. 5, for each pair of primers. It ispossible to observe between 1 and 11 amplification peaks, eachcorresponding to a particular size for the CDR3 region and eachseparated by three nucleotides. This analysis can be further refined ifa primer specific for a Jβ segment is used instead of the common Cβprimer. As illustrated in FIG. 6, analysis of the distribution of thevarious CDR3 lengths makes it possible to identify possible oligoclonalexpansions.

The Reperturb software makes it possible to compare the CDR3 lengthdistribution in a given sample with that obtained in a referenceindividual (Gaussian profile, not altered). Each CDR3 length analyzed bythe Immunoscope® software is translated into a probability ofdistribution, taking into account the area under the curve for eachpeak. For each one of them, the difference in absolute value between thesample (En) and the reference profile (Cn) makes it possible to obtainthe percentage alteration of the sample relative to a Gaussian profile.This analysis therefore makes it possible to specify the level ofalteration observed on an Immunoscope® profile. The variations obtainedfor each CDR3 expansion in a Vβ family will therefore be expressed aspercentage alteration of a peak within this family. The sum of thealterations in all the Vβ families makes it possible to estimate thepercentage of overall alteration of the TCR Vβ chain in a T cellpopulation.

It is then necessary to perform the quantification of the Vβtranscripts, which can be approached using several techniques. In thecase in point, the real-time quantitative PCR method can be used. Thismethod, based on the use of the TaqMan® technology, can use the programknown as “ABI Prism 7700 Sequence Detection System” which makes itpossible, inter alia, to detect and measure the fluorescence emitted bythe binding of a label (Sybr®Green) to double-stranded DNA molecules.The level of fluorescence, which is directly proportional to the amountof the product in a well, is collected in the course of eachamplification cycle. In parallel with the samples, a standard,corresponding to a target sequence dilution range, can be used and willmake it possible to establish a standard curve, which is used to deducethe amount of the target in each sample. In order to eliminateexperimental variations between the various samples, these values arerelated to the measurement of the housekeeping gene HPRT.

In order to improve the specificity and the sensitivity of the PCR, theenzyme called AmpliTaq Gold® DNA polymerase is used. A modification inthis enzyme makes it active only at high temperature, a temperature atwhich the DNA is completely denatured. In order to avoid a contaminationwith other PCR products, AmpErase® uracil-N-glycosylase (UNG) is addedto the reaction mixture at the time of assaying. This enzyme, which isonly active below 60° C., acts by hydrolyzing the uracil bridges insingle- or double-stranded DNA containing uracil bases and has noactivity on DNA containing thymidines. These uracil bridges aregenerated by the introduction of dUTP bases into the reaction mixture.During the amplification, an initial step of 2 minutes at 50° C.activates this amperase and makes it possible to eliminate possiblecontaminants derived from the preceding amplification.

The standards are prepared from reverse-transcribed RNA originatingeither from splenocytes from a naïve animal, or from T lymphocytesderived from the blood of healthy individuals, in humans. For thepreparation of each Vβ standard, the amplification is carried out in aconventional thermocycler. The PCR products are then loaded onto anagarose gel and the band of interest is extracted. The concentration ofthe PCR product is estimated through the OD₂₆₀ value and then, as afunction of the molecular mass, the number of copies per ml iscalculated. Dilutions of each Vβ, Cβ or HPRT standard at 10⁷, 10⁶, 10⁵,10⁴, 10³ and 10² copies per well are then prepared in order to obtain aconcentration range which covers that of the samples.

In order to validate each standard, a second PCR is carried out, thistime in the ABI PRISM 7700 Sequence Detector in the presence of theSybr®Green fluorescent label. The PCR products are then loaded onto anagarose gel, which makes it possible to control the decreasing reductionin each band as a function of the dilution and also the size of the PCRproduct. In parallel, the increase in fluorescence intensity, which isfollowed as a function of time, is linked to the number of copies of DNAcontained in each dilution. This makes it possible to verify theaccuracy of the dilutions for the standard, the PCR yield and theabsence of contamination.

Once validated, these standards make it possible to deduce the number ofcopies of the target in each sample, tested in duplicate, by relatingthe level of fluorescence to the standard straight line. This straightline is only taken into account if the efficiency of the PCR is close to100% (slope=−3.3) and if the coefficient of correlation is greater than0.95.

It is then a question of combining the qualitative and quantitativeanalyses so as to give a quantitative dimension to the alterations inthe CDR3 length distribution. This information can make it possible toevaluate the representativeness of an oligoclonal expansion in a givenpopulation and can also make it possible to visualize modifications inthe repertoire which are not associated with alterations in the CDR3region.

The invention will preferably use a computer program developed in orderto obtain an image containing all the information relating to therepertoire studied. The Matlab® software can be used for this purposesince it thus enables the flexible management of a very large volume ofdata and an imaging which is sufficiently clear and informative. Foreach Vβ family, the percentage alteration relative to the Gaussianprofile is measured by the Gorochov method. By virtue of the Matlab®software, these values are combined with the first (quantitative) dataso as to obtain a 3D representation illustrating both the qualitativeand quantitative variations in the Vβ transcriptome, in which the heightof the peaks illustrates the amount of corresponding Vβ transcripts,while the percentage alterations are represented by a color code.

On the graph in FIG. 9, the following appear:

-   -   along the x-axis, each one of the Vβ families;    -   along the y-axis, the quantitative data (i.e. the relative size        of each Vβ family);    -   along the Z-axis, the various possible lengths of the CDR3; and    -   according to a color code, a visualization of the alterations in        the repertoire relative to a Gaussian profile.

The color code can comprise a series of colors, for example from greento red in the direction of increasing alteration, i.e. with increasingdistance from 0% alteration. In the case in point, it will involvevarious levels of gray, the gray becoming darker and darker as thealteration increases.

Thus, the Vβ transcripts, which may or may not be altered, are expressedaccording to a three-dimensional graph exhibiting, along the x-axis,each Vβ family and, along the y-axis, the quantitative data obtained byTaqMan (TaqMan PCR for Cβ and for each Vβ mRNA species). The percentagealteration for each Vβ relative to a Gaussian profile is visualizedaccording to a color code (an alteration “range” corresponds to eachcolor).

The method will be implemented by means of an installation which makesit possible to analyze the T lymphocyte receptors of an organism,illustrated diagrammatically in FIG. 10. This installation will compriseautomated means for amplifying the CDR3 region of each of the Vβ genesof the T lymphocyte receptors associated with the organism (obtainingthe second values), and for quantifying each Vβ gene (first data). Itmay, for example, be a machine (2) such as the TaqMan® device, whichmakes it possible, by virtue of following the amplifications in realtime, to quantify the Vβ transcripts as they are amplified: thequantitative values thus obtained make it possible to compare amounts oftranscripts of a certain Vβ family with amounts of transcripts ofanother Vβ family (example of device: ABI Prism 7900 from the companyApplied Biosystems).

The installation will also comprise automated means for measuring therelative amounts of each length of the CDR3 region in each Vβ family andthe ratio of these amounts to predetermined values, so as to obtainsecond data. It may be a sequencer (4) which makes it possible todetermine the relative part of each transcript having a particular sizefor CDR3 in a given Vβ family, but which does not make it possible tocompare the Vβ families with one another: (for example, the Magabacecapillary sequencer from the company Amersham pharmacia biotech).

Finally, the installation will comprise automated means forrepresenting, in a four-variable diagram, the first and second data as afunction of the Vβ genes and of the length of the CDR3 regions. It maybe a computer (6) controlled by a program developed, for example, usingthe abovementioned Matlab® software. The development of such a programin itself is within the scope of those skilled in the art and will notbe detailed here. Optionally, this program will be able to control othersteps in the progression of the method, or even the entire method. Thediagram will be represented on an information medium, such as a computerscreen or a paper medium (8).

This study can be carried out in the course of the kinetics of an immuneresponse in various situations.

In this context of implementation, as in others, the invention makes itpossible to carry out more thorough analyses than with the methods ofthe prior art. The invention makes it possible to obtain a general and athree-dimensional view of the size of the pool of T cells using aparticular Vβ rearrangement and therefore makes it possible to determinethe quantitative importance of the Vβ families selected during theimmune response.

Thus, with reference to FIG. 11, which gives an example of theappearance of a diagram not associated with particular biologicalcircumstances, it appears that some families, although highly alteredrelative to the Gaussian profile, are only weakly represented at thequantitative level and therefore do not appear to have a predominantrole (example: family x). The invention also makes it possible todifferentiate a resting situation from a situation in which the T cellsare activated in a polyclonal manner. Specifically, the graph shown inFIG. 12 is derived from a model in which T cells are activated in apolyclonal manner, and a very strong mobilization of certain Vβ familieswhich, nevertheless, exhibit a Gaussian distribution for the CDR3lengths (which corresponds to 0% alteration) is then observed. Thus,this technique makes it possible, inter alia, to visualize polyclonal.activations (ConA, superantigen, direct presentation pathway) whichwould not have been detected by conventional tools. It is therefore apowerful means for monitoring the changes in the repertoire in thecourse of various immune responses.

In fact, the graph is derived from a model in which T cells areactivated in vitro, and a very strong mobilization of certain Vβfamilies is observed. On this same scale, a control sample would give agreen (light gray) profile since all the families exhibit a CDR3 lengthdistribution which is Gaussian (which corresponds to 0% alteration) andwhich is flat since all the Vβ families are weakly represented. It istherefore a powerful means for monitoring the changes in the repertoirein the course of various immune responses.

FIG. 13 illustrates the diagram resulting from a second embodiment ofthe invention. The invention is here used in the context of a studycarried out on patients who are suffering from melanoma and who havebeen vaccinated. The Vβ13 family appears to be particularly involved inthe anti-melanoma response insofar as a peak appears which is red (darkgray), and therefore distant from the Gaussian profile characteristic ofa normal response, and quantitatively important. Another family appearsto have a less important role: the Vβ23 family, which is perturbed butrelatively unimportant in quantitative terms. In addition, an unresolvedpeak appears in the region of the first Vβ families. This unresolvedpeak might correspond to an effect of the vaccination. Further studieswill make it possible to determine this.

Described below is another embodiment in which the method of theinvention is completed by a step consisting of isolating the clonesexhibiting altered Vβ families.

The specific alterations in the repertoire which are detected using theinvention are, most commonly, not detectable by conventional techniquesof flow cytometry. In fact, while the invention makes it possible todetect an alteration and an accumulation of transcripts for a given Vβfamily, flow cytometry only makes it possible to detect themembrane-bound expression of the Vβ protein. Now, surface proteinexpression is transient and does not make it possible to detect amodification in the transcriptional activity of a cell at a precisemoment.

As has been seen, the invention thus makes it possible to detect amodification in transcriptional activity for a given family. Once thefamily has been detected, it is possible to extract it from the rest ofthe lymphocyte population in order to study the specific characteristicsthereof (phenotype, activation, etc.). If these characteristics werestudied in the total population, they would be masked by the sum of thecharacteristics of all the other T cell populations.

The present method of representation therefore makes it possible toidentify particular populations involved in the immune response studied.These T cells, bearing a TCR composed of a particular Vβ family, canthen be isolated by flow cytometry using anti-Vβ antibodies, forexample, which makes it possible to characterize the clones bearingspecific (qualitative and/or quantitative) alterations in the TCRβ-chain repertoire.

After implementation of the invention as set out in the precedingembodiments, including the step for producing the diagram, the cellsexpressing the various Vβ families characterized by oligoclonalexpansions and/or large amounts of messenger RNAs are extracted by flowcytometry using antibodies directed against the various Vβ families.

Thus, each Vβ family before sorting represents less than 5% of the Vβrepertoire. After sorting, it is possible to work on a given populationwhich is very pure since the purity obtained is greater than 98%.

Several successive rounds of sorting are possible and therefore severalT cell clones can be isolated from the same cell extract since, afterselection of a particular T cell population, the remaining populationcomprises all the other T cell types. This same cell extract which hasmade it possible to isolate a first T cell population may therefore alsoserve as a basis for extracting another Vβ family. The possibilities andcontributions of this technique are therefore very considerable andimprove the basic technique of the invention. Once the number of cellsextracted has been determined, the cells are used for phenotypic andtranscriptional studies.

These studies are fundamental since they will make it possible todetermine the role of a lymphocyte clone in a given situation. Thisdetermination of the role of T cell clones is very important insofar as,if the clone identified proves to have a cytotoxic role (viruses such asHIV, CMV or EBV; autoimmune diseases), it will be necessary to inhibitit in the context of immunotherapeutic strategies. Conversely, if thisclone proves to have a protective role (transplantation,immunosuppressor treatments), it should be stimulated in order to obtaina beneficial effect.

To implement this technique, a flow cytometer may be used which makes itpossible to isolate the T cells revealed by the technology according tothe invention, after labeling of the T cell populations considered withan anti-Vβ antibody, such as that marketed under the name “Facscalibur”by the company Beckton Dickinson.

As illustrated by FIG. 14, the invention also relates to a method foridentifying, in particular T lymphocyte populations, genes, moleculesand mechanisms involved in physiopathological processes.

The method of analysis according to the invention is carried out and theT cell populations for which the TCR transcripts are stronglyrepresented among all the transcripts of the total T cell population canbe isolated, and the phenotype of these cells can be compared withrespect to the phenotype of the cells of the general population, thusmaking it possible to demonstrate the pathways of response to stimuli.

Of course, many modifications may be introduced into the inventionwithout departing from the context of the attached claims. Thus, on thediagram, the variable represented by differences in local appearance ofthe diagram may be one which is different from that corresponding to thesecond data.

The difference in local appearance may be illustrated other than bycolors or shades of gray. It may, for example, be various types ofhatchings or other types of signs.

The invention also relates to each primer for amplifying the CDR3regions, as indicated in the sequence listing.

1. A method for analyzing the activation and mobilization of T cellsthrough an analysis of the T lymphocyte receptors of an organism,characterized in that it comprises the steps consisting in: determiningthe number of transcripts for each Vβ gene, so as to obtain first data;amplifying the CDR3 region of the T lymphocyte receptors for each of theVβ genes associated with the organism; separating the various lengths ofthe CDR3 of the T lymphocyte receptors for each of the Vβ genesassociated with the organism and estimating the proportion oftranscripts for each length of the CDR3 in each Vβ family, so as toobtain second data; representing, in a four-variable diagram, the firstand second data as a function of the Vβ genes and of the length of theCDR3 regions. 2-11. (canceled)
 12. The method as claimed in claim 1,characterized in that at least one of the variables is representedwithout being associated with a dimension in space.
 13. The method asclaimed in claim 12, characterized in that the variable which is notassociated with a dimension in space corresponds to the second data. 14.The method as claimed claim 1, characterized in that at least one of thevariables is represented by differences in local appearance of thediagram.
 15. The method as claimed in claim 14, characterized in thatthe differences in local appearance are color differences.
 16. Themethod as claimed claim 1, characterized in that the first data arerepresented in the vertical direction.
 17. The method as claimed claim1, characterized in that it comprises, after determination of the firstand second data, the steps consisting in choosing a Vβ gene as afunction of the results from the first two steps, and in extracting fromthe total T cell population the T cells bearing a TCR consisting of thispreselected Vβ family.
 18. The method as claimed in claim 1,characterized in that at least some of the steps are carried out in anautomated fashion.
 19. A computer program for analyzing the T lymphocytereceptors of an organism, characterized in that it is capable ofcontrolling the implementation of at least the representation step ofthe method as claimed in claim
 1. 20. An installation for analyzing theT lymphocyte receptors of an organism, characterized in that itcomprises: automated means for amplifying the CDR3 region of the Tlymphocyte receptors for each of the Vβ genes associated with theorganism; automated means for measuring the amounts of each Vβ gene, soas to obtain first data; automated means for measuring, for each Vβgene, the relative amount of each CDR3 region with respect to all theCDR3 regions associated with a Vβ gene, so as to obtain second data; andautomated means for representing, in a four-variable diagram, the firstand second data as a function of the Vβ genes and of the length of theCDR3 regions.
 21. An information medium exhibiting a diagram analyzingthe T lymphocyte receptors of an organism, characterized in that itcomprises the following four variables: the Vβ genes associated with theorganism; the length of the CDR3 regions of the T lymphocyte receptors;first values representing the amount of transcripts of each Vβ gene inthe form of relative or absolute amount; second data representing, foreach of the lengths of the CDR3 region in each Vβ gene, the differencebetween the proportion of transcripts corresponding to a CDR3 region ofa Vβ gene in a given sample compared to the transcripts corresponding tothe same CDR3 region of the same Vβ gene in a reference sample which isa sample exhibiting a Gaussian distribution for CDR3 lengths.